The overall goal of this research is to understand the role(s) of cell surface glycoproteins, particularly sialomucins, in mammary cancer. Sialomucins have been implicated in the escape of tumors from immune destruction and in metastasis and provide potentially important markers for immunological diagnosis, prognosis, and therapy. The model system being studied is the cell surface sialomucin complex of metastatic 13762 rat mammary adenocarcinoma ascites cells, composed of the sialomucin ASGP-1 and its associated N-linked glycoprotein ASGP-2. Previous structure and biosynthetic investigations make ASGP-1 one of the best studied tumor cell surface sialomucins known. It is a particularly useful model for the study of sialomucin complex structure, biosynthesis and expression because of its abundance in the ascites cells (1% of total cell protein) and the variation in its expression with growth conditions of the tumors. Recent biosynthesis and structure studies suggest the following. 1) ASGP-1 and ASGP-2 are synthesized as a single large precursor pSMC-1, which is cleaved and immediately O-glycosylated. 2) Maturation of ASGP-1 and expression of cell surface "incomplete" oligosaccharides involve addition of new oligosaccharides during a recycling process after ASGP-1 has reached the cell surface, a 2nd pathway for O-glycosylation. 3) ASGP-2 is related to cell surface receptors and growth factors which contain the EGF/notch sequence. The sites for pSMC-1 cleavage and for O-glycosylation will be investigated by kinetic analyses, temperature dependence studies, cell fractionations and inhibitor analyses. The role and mechanism of recycling in sialomucin biosynthesis will be investigated by examining the kinetics of glycosylations and of recycling of cell surface ASGP-1 using a biotinylation procedure. The localizations of the recycling process and of the enzymes involved in adding new oligosaccharides during recycling will be investigated by cytochemistry and by enzyme analyses of isolated, sealed, cell surface microvilli, respectively. The role of processing in the localization of sialomucin antigens in human breast tumor cell lines will be investigated using pulse-chase kinetics and recycling analyses. The relationship of pSMC-1 to ASGP-2 and ASGP-1, of ASGP-2 to other cell surface molecules and of ASGP-1 to other sialomucins will be studied by cDNA cloning and sequencing. The relationship of the expression of the sialomucin complex to tumor progression will be examined using immunological and cDNA probes. These combined studies should yield valuable information for elucidating aspects of the biosynthesis and expression of tumor cell surface sialomucin complex components which are important to understanding their roles in tumor biology.